iPSC & iPSC derived cells- Values and Applications in drug ... · important in the early drug discovery processes • Multiple, biologically relevant targets and pathways are simultaneously

Discovery Sciences | Reagents & Assays

Mei DingIPSC&A (iPSCs, Primary cells, Stem Cells & Assays)

Discovery SciencesAstraZeneca

iPSC & iPSC derived cells-Values and Applications in drug discovery

Items

• Introduction-iPSC values and applications

• iCells Cardiomyocyte validation examples

• Future opportunities and challenges

2 Discovery Sciences | Reagents & Assays

3 Discoverynn Sciences | Reagents & Assays

Drug discovery processes• Use of biologically relevant cells in physiological in vitro assays is very important in the early drug discovery processes• Multiple, biologically relevant targets and pathways are simultaneouslyinterrogated in biologically relevant cells

Biologically relevant cells• Human primary cells• Pluripotent stem cells derived cells

• ESC derived cells• iPSC derived cells

• Adult stem cells (ASC)• etc

Target Validation

Lead Generation

Lead Optimization

Pre-clinical Development

LGID CSIDLOID

Clinical Studies

FTIM


Human primary cells • Difficult to obtain for certain cell types• Limited proliferative capacity• Challenge to maintain the in vivo phenotype in culture• Large batch variations

Human primary cells, ESCs & ASCsLimitations

hES cells • Limited access• Significant ethical and political debates

Human adult stem cells • Limited access• Often limited in their proliferative ability and can become only the cell type(s) of the organ where they come from

5Discovery Sciences | Reagents & Assays

Patients Patients

Screening

”Disease in a dish”

Differentiatedcells

iPS cell production

iPScells

Somaticcell

Cell expansion

iPSCcharacterisationDrugs

Reprogram-ming factors

• Gene expression • Epigenetics• DNA fingerfrinting• Karyotyping• Pluripotency

[Embryoid bodies(EB), Teratomas, directeddifferentiation]

Human iPSC & iPSC derived cells

iPSCgeneration

• Clone selection• Expansion • Banking

Reprogramming of human adult somatic cells to become hESC-like induced

pluripotent stem cells (iPSCs). hiPSCs have the ability to divide indefinitely


Patients Patients

Screening


Differentiatedcells

iPS cell production

iPScells

Somaticcell

Cell expansion

iPSCcharacterisationDrugs

Reprogram-ming factors

• Gene expression • Epigenetics• DNA fingerfrinting• Karyotyping• Pluripotency

[Embryoid bodies(EB), Teratomas, directeddifferentiation]

iPSC


iPSCgeneration

In vitro differentiationto tissue specific cell

types

• Clone selection• Expansion • Banking

iPSCs largely resemble hESCs and can be differentiated in vitro to tissue

specific cell types

Discovery Sciences | Reagents & Assays

Human iPSC directed differentiation in vitro

7

Patients

Screening


Differentiatedcells

iPScells

iPSCDrugs

In vitro differentiation to tissuespecific cell types

Assaydevelopment

iPS Cells

Endoderm

Liver

Intestine

Pancreas

Mesoderm

Lung

Heart

Fat

Ectoderm

Brain

Nervoussystem

Eye

• Possibilities

iPSCs have the potential to become all cell types of the body , can therefore

serve as a potential unlimited ex vivo source of human derivatives


Human iPSCs & iPSC derived cells in Drug discovery

• Target ID / target validation• Disease in vitro models• Phenotypic screening• 2nd assays & MOA• Efficacy• Safety / Toxicity• DMPK• etc…

• Easy to handle• Scalable• Stable & Robust• hiPSC directed In vitro differentiation

to tissue specific cell types• Functional• Cost effective

hiPSC advantages hiPSC applications

Target Validation

Lead Generation

Lead Optimization


LGID CSIDLOID

Clinical Studies

FTIM

9Discovery Sciences | Reagents & Assays

iPSCgeneration

AZ iPSC capabilities (small-scale)

Scale up iPSCcharacterisation

Pluripotent QC of hiPSC

Grafting iPSCunder kidneycapsule in SCID mouse

Differentiatedcells

iPSC directeddifferentiation

in vitro

Accessing External World-leading Expertise in stem cell technology

Cellular Dynamics International (CDI) & AZ (CVMD, GSA, DMPK and DS)

• On-going collaboration with access to the iCELL products and other products for use in the safety, discovery and regenerative medicine programs

Discovery Sciences | Reagents & Assays10

Examples of AZ on-going iCells validation

• Use of human iPSCs in toxicity testing

• Use of human iPSCs in therapy areas - Cardiomyocyte proliferation assay


In vitro safety tools

12 Joanne Bowes | April 2013 Innovative Medicines | Global Safety Assessment

Impact

• Biochemical and cellular assaysCell lines

• Recombinant human screens, cardiac ion channel screens,

• Transporter assays• Secondary Pharmacology

screens

Molecular

• Cardiomyocytes• Hepatocytes

Primary cells

• Isolated heart• Brain sliceTissue

Will human stem cell derived cells offer a scientific or technical advantage?

Where we are now Where we need to be

Primary short-term objectiveRobust screens predictiveof human toxicity

Measured bySensitivitySpecificityPredictivityRobustness

Cost:benefit

Technology availability

Use of iCells in Safety Assessment

Innovative Medicines | Global Safety Assessment

Applied in three modes

1. Front loading of in vitro ‘black box’ and molecular assays prior to toxicology studies

• Routine screening within the design make test cycle

2. Bespoke target safety

3. Investigative toxicology• De-risk follow up compound series• Provide mechanistic understanding to observed in vivo toxicology

In vivo models

In vitro models

Lead Generation

Lead Optimization


LGID CSIDLOID

Clinical Studies

FTIM

13

Potential use of iCell Products for Front loading

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Cardiotoxicity

1. Front loading - in vitro ‘black box’ and molecular assays in medium –high throughput format

Hepatotoxicity

Chronotropes Beat rate

Inotropes Beat rate/amplitudeCa2+ flux

ECG modulation APDIon channel inhibition

Pathology change High Content Biology (HCB)

Dysfunction Measure

Pathology change High Content Biology (HCB)

Innovative Medicines | Global Safety Assessment

http://www.alphalabs.co.uk/cms/site/images/Liver-illustration.jpg

http://3.bp.blogspot.com/_CarNcodpCMA/TSxSgsS6uSI/AAAAAAAAKY4/nWtd1ERnLrU/s1600/heart-vintageanatomy-graphicsfairy007red.jpg

Use of in vitro models in safety assessment

Applied in three modes

1. Front loading of in vitro ‘black box’ and molecular assays prior to toxicology studies

• Routine screening within the design make test cycle

2. Bespoke target safety

3. Investigative toxicology• De-risk follow up compound series• Provide mechanistic understanding to observed in vivo toxicology

In vivo models

In vitro models

Lead Generation

Lead Optimization


LGID CSIDLOID

Clinical Studies

FTIM

Innovative Medicines | Global Safety Assessment15

Target Safety and Investigate toxicologyPotential use of iCell in Impedance assay to explore target safety and human CV safety risks

• Using impedance to measure cell function (beating)

• Using siRNA and tool compounds to explore target versus off-target toxicity

• Data shown was from rat neonatal cardiomyocytes

• Moving to iCells: focus on human safety risks!

beat amplitude

beat rate

Time (sec)

Cel

l Ind

ex

RTCA Cardio impedance instrument quantifes cardiomyocyte beating

*

***

**

Target contributes to CV toxicity seen in vivo

A B

9 inhibitors

•Beat IC50 & kinase inhibition IC50’s not correlated•siRNA has negligible effect on beating

CV toxicity in vivo unlikely due to target Lamore & PetersAstraZeneca, Boston

Kinase-A

Kinase-B

CtrlsiRNA

K1 K2 K1/2

K1 K2 K3 K4

Ctr

lsiR

NA

•Kinase inhibitors ↓beat amplitude•Potencies correlate with kinase affinities•siRNA ↓ amplitude

K1-

4


Proliferation assay for cardiac regeneration phenotypic screening

Background: Unmet medical need for post-MI and heart

failure patients

Fundamental problem: Loss of functional cells

Goal: Regenerate functional myocardium

Task: To identify noval targets or chemistry that can induce

cardiomyocyte proliferation for the treatment of post-MI

cardiac dysfunction and heart failure

Phenotypic screening approach


• The phenotypic screening was the most successful approach for first-in-class drugs discovered between 1999 and 2008

• Idealy use native human cells (multiple, biologically relevant targets and pathways are simultaneously interrogated)

CDI iCell Cardiomyocyte

• Measure the desired biological effect (activity might be translated to human disease more effectively than that in target-based screens):

Cardiomyocyte proliferation


Challenge 1: Mature CMsdon’t proliferate and iPSderived CMs proliferateslowly. Potential smallassay window

Total DNA

ATP

Edu DNA

synthesis

Ki67 Prolif. marker

Nuclearnumber

Cell number

Potential samll assaywindow due to slow CMproliferation

An ideal readout,but difficult for CMSensitive, an early

indication of cellproliferation

Edu High ContentImage assay

Assay options and challenges

Challenge 2: CMs areheterogenous (mono-, bi-,multi-nuclei). Can’t usenuclei to count cell number.Need a good membranemarker to do cellsegmentation.

Edu HCS assay


Load cells

with Edu

Alexa Flour azide

CuSO4

Fix

cells

Staining of nuclei

& other markersImage

Edu staining

Assay parameters optimized

• Different assay media

• Coating: Gelatin, Collagen, CellBind surface

• Edu loading conc

• Cell density

• With or without serum starvation

• Compound treatment period

Plate cells (3.5k c/w)

384-w

Exchange to CDI M M

2-3 days

Add cpds & Edu in

Assay M

Fix cells

2 days 2 days

Staining& Image



Medium supplement effect on proliferationMedium-BMedium-A

Edu staining

CTnT staining

10x

• A basal CM proliferation in medium-A, 6-7% of nuclei were Edu+ with 2-d Edu loading

• Medium-B enhanced CM proliferation significantly

• bFGF in medium-A gave slightly less effect than medium-B alone

0.0

5.0

10.0

15.0

20.0

25.0

Medium-A Medium-B bFGF in medium-A

% E

du +

nuc

lei

Tool compound effect on CM proliferation


BIO (GSK3beta inhibitor) bFGF

EC50 = 160 nM EC50 = 120 pg/ml

BIO

Concentration (µM))0.1 1

%Ed

u+

nucl

ei(n

orm

aliz

edda

ta)

-20

0

20

40

60

80

100

0.03 0.1 0.3 1 Concentration (µM)% E

du +

nuc

lei(

norm

aliz

edda

ta)

bFGF

Concentration (ng/ml)0.1 10

%E

du+

nucl

ei(n

orm

aliz

edda

ta)

-20

0

20

40

60

80

100

120

0.1 1 10 100Concentration (ng/ml)% E

du +

nuc

lei(

norm

aliz

edda

ta)

• GSK3beta inhibitor BIO and bFGF induced dose dependent increase in the number

of Edu+ nuclei, an early indication of CM proliferation


Future opportunities• “Disease in a dish” - Disease modeling

• Patient-derived iPSCc• Genome editing to introduce disease variant

• “Patient in a dish” - Personalised medicine • “Clinical trials in a dish” - Patient variation • Opportunity to increase understanding of clinical trial out-come by

generating iPS cells from stored blood samples• Regenerative therapy


Challenges• Control differentiation to a fully mature cell (ex. CM)• Reach a highly pure, homogeneous and mature population• Reach full ‘adult’ maturation when cultured in vitro.

Acknowledgements


This is cross-function activity

Discovery SciencesHenrik Andersson, Gabriella Brolén, Elke Ericson, Philip Worthington, Sam Peel, Anna Jonebring, Anders Lundin, Anette Persson-Kry, Björn Löwenadler

CVMDLauren Drawley, Qing-Dong Wang, Britt-Marie Kihlberg, Alan Sarbish, Bruno Becker

GSAHelen Garside, Joanne Bowes, Amy Pointon, Clay Scott, Matt Peters, Sara Lamore


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iPSC & iPSC derived cells- Values and Applications in drug ... · important in the early drug discovery processes • Multiple, biologically relevant targets and pathways are simultaneously